Broadly Applicable Bispecific Linker Approach to Noncovalently Target Therapeutic Nanoparticles to Tumor Cells Expressing Carcinoembryonic Antigen.

Design strategies that lead to a more focused in vivo delivery of functionalized nanoparticles (NPs) and their cargo can potentially maximize their therapeutic efficiency while reducing systemic effects, broadening their clinical applications. Here, we report the development of a noncovalent labeling approach where immunoglobulin G (IgG)-decorated NPs can be directed to a cancer cell using a simple, linear bispecific protein adaptor, termed MFE23-ZZ. MFE23-ZZ was created by fusing a single-chain fragment variable domain, termed MFE23, recognizing carcinoembryonic antigen (CEA) expressed on tumor cells, to a small protein ZZ module, which binds to the Fc fragment of IgG. As a proof of concept, monoclonal antibodies (mAbs) were generated against a NP coat protein, namely, gas vesicle protein A (GvpA) of Halobacterium salinarum gas vesicles (GVs). The surface of each GV was therapeutically derivatized with the photoreactive agent chlorin e6 (Ce6GVs) and anti-GvpA mAbs were subsequently bound to GvpA on the surface of each Ce6GV. The bispecific ligand MFE23-ZZ was then bound to mAb-decorated Ce6GVs via their Fc domain, resulting in a noncovalent tripartite complex, namely, MFE23.ZZ-2B10-Ce6GV. This complex enhanced the intracellular uptake of Ce6GVs into human CEA-expressing murine MC38 colon carcinoma cells (MC38.CEA) relative to the CEA-negative parental cell line MC38 in vitro, making them more sensitive to light-induced cell killing. These results suggest that the surface of NP can be rapidly and noncovalently functionalized to target tumor-associated antigen-expressing tumor cells using simple bispecific linkers and any IgG-labeled cargo. This noncovalent approach is readily applicable to other types of functionalized NPs.

Development of a Small-Footprint 50 MHz Linear Array: Fabrication and Micro-Ultrasound Imaging Demonstration.

Compact high-frequency arrays are of interest for clinical and preclinical applications in which a small-footprint or endoscopic device is needed to reach the target anatomy. However, the fabrication of compact arrays entails the connection of several dozens of small elements to the imaging system through a combination of flexible printed circuit boards at the array end and micro-coaxial cabling to the imaging system. The methods currently used, such as wire bonding, conductive adhesives, or a dry connection to a flexible circuit, considerably increase the array footprint. Here, we propose an interconnection method that uses vacuum-deposited metals, laser patterning, and electroplating to achieve a right-angle, compact, reliable connection between array elements and flexible-circuit traces. The array elements are thickened at the edges using patterned copper traces, which increases their cross-sectional area and facilitates the connection. We fabricated a 2.3 mm by 1.7 mm, 64-element linear array with elements at a 36 µm pitch connected to a 4 cm long flexible circuit, where the interconnect adds only 100 µm to each side of the array. Pulse-echo measurements yielded an average center frequency of 55 MHz and a -6 dB bandwidth of 41%. We measured an imaging resolution of 35 µm in the axial direction and 114 µm in the lateral direction and demonstrated the ex vivo imaging of porcine esophageal tissue and the in vivo imaging of avian embryonic vasculature.

Adaptation of a Clinical High-Frequency Transrectal Ultrasound System for Prostate Photoacoustic Imaging: Implementation and Pre-clinical Demonstration.

OBJECTIVE: High-frequency, high-resolution transrectal micro-ultrasound (micro-US: ≥15 MHz) imaging of the prostate is emerging as a beneficial tool for scoring disease risk and accurately targeting biopsies. Adding photoacoustic (PA) imaging to visualize abnormal vascularization and accumulation of contrast agents in tumors has potential for guiding focal therapies. In this work, we describe a new imaging platform that combines a transrectal micro-US system with transurethral light delivery for PA imaging. METHODS: A clinical transrectal micro-US system was adapted to acquire PA images synchronous to a tunable laser pulse. A transurethral side-firing optical fiber was developed for light delivery. A polyvinyl chloride (PVC)-plastisol phantom was developed and characterized to image PA contrast agents in wall-less channels. After resolution measurement in water, PA imaging was demonstrated in phantom channels with dyes and biodegradable nanoparticle contrast agents called porphysomes. In vivo imaging of a tumor model was performed, with porphysomes administered intravenously. RESULTS: Photoacoustic imaging data were acquired at 5 Hz, and image reconstruction was performed offline. PA image resolution at a 14-mm depth was 74 and 261 µm in the axial and lateral directions, respectively. The speed of sound in PVC-plastisol was 1383 m/s, and the attenuation was 4 dB/mm at 20 MHz. PA signal from porphysomes was spectrally unmixed from blood signals in the tumor, and a signal increase was observed 3 h after porphysome injection. CONCLUSION: A combined transrectal micro-US and PA imaging system was developed and characterized, and in vivo imaging demonstrated. High-resolution PA imaging may provide valuable additional information for diagnostic and therapeutic applications in the prostate.

Superharmonic and Microultrasound Imaging With Plane Wave Beamforming Techniques.

Superharmonic contrast imaging (SpHI) suppresses tissue clutter and allows high-contrast visualization of the vasculature. An array-based dual-frequency (DF) probe has been developed for SpHI, integrating a 21-MHz, 256-element microultrasound imaging array with a 2-MHz, 32-element array to take advantage of the broadband nonlinear responses from microbubble (MB) contrast agents. In this work, ultrafast imaging with plane waves was implemented for SpHI to increase the acquisition frame rate. Ultrafast imaging was also implemented for microultrasound B-mode imaging (HFPW B-mode) to enable high-resolution visualization of the tissue structure. Coherent compounding was demonstrated in vitro and in vivo in both imaging modes. Acquisition frame rates of 4.5 kHz and 187 Hz in HFPW B-mode imaging were achieved for imaging up to 21 mm with one and 25 angles, respectively, and 3.5 kHz and 396 Hz in the SpHI mode with one and nine coherently compounded angles, respectively. SpHI images showed suppression of tissue clutter prior to and after the introduction of MBs in vitro and in vivo. The nine-angle coherently compounded 2-D SpHI images of contrast-filled flow channel showed a contrast-to-tissue ratio (CTR) of 26.0 dB, a 2.5-dB improvement relative to images reconstructed from 0° steering. Consistent with in vitro imaging, the nine-angle compounded 2-D SpHI of a Lewis lung cancer tumor showed a 2.6-dB improvement in contrast enhancement, relative to 0° steering, and additionally revealed a region of nonviable tissue. The 3-D display of the volumetric SpHI data acquired from a xenograft mouse tumor using both 0° steering and nine-angle compounding allowed the visualization of the tumor vasculature. A small vessel visible in the compounded SpHI image, measuring around [Formula: see text], is not visualized in the 0° steering SpHI image, demonstrating the superiority of the latter in detecting fine structures within the tumor.

Fine Pitch Flexible Printed Circuit Board Patterning for Miniaturized Endoscopic MicroUltrasound Arrays.

Microultrasound (micro-US) has become an invaluable tool for preclinical research and in emerging applications in clinical diagnosis and treatment guidance. Several such applications can benefit from arrays with a small footprint and endoscopic form factor. However, critical challenges arise in making electrical connections to array elements in such spatial constraints. In this work, we describe a method to pattern a high-density flexible circuit cabling on a copper-on polyimide film, using laser ablation of a polymer resist and wet etching, and then demonstrate a connection to a micro-US array. We investigate laser ablation process parameters and evaluate the ability to consistently pattern continuous copper traces. A minimum 30- [Formula: see text] pitch was achieved with 5- [Formula: see text]-wide electrode lines, and continuity of a 5-m-long trace is demonstrated. A flexible circuit with 30-mm-long traces with 30- [Formula: see text] line and 30- [Formula: see text] space before fan-out was fabricated to connect in an interleaved manner to a 32-element array with 30- [Formula: see text] element pitch. Metal deposition and laser ablation were used to connect and pattern the element electrodes to the copper traces of the flexible circuit. Electrical and acoustic measurements show good yield and consistent impedance across channels. Element pulse-echo tests demonstrated device functionality; the two-way pulse had 43-MHz center frequency and 40% fractional bandwidth (-6 dB). The proposed manufacturing methods facilitate the prototyping and fabrication of flexible endoscopic or small-footprint micro-US devices.

Acoustic Molecular Imaging Beyond the Diffraction Limit In Vivo.

Ultrasound molecular imaging (USMI) is a technique used to noninvasively estimate the distribution of molecular markers in vivo by imaging microbubble contrast agents (MCAs) that have been modified to target receptors of interest on the vascular endothelium. USMI is especially relevant for preclinical and clinical cancer research and has been used to predict tumor malignancy and response to treatment. In the last decade, methods that improve the resolution of contrast-enhanced ultrasound by an order of magnitude and allow researchers to noninvasively image individual capillaries have emerged. However, these approaches do not translate directly to molecular imaging. In this work, we demonstrate super-resolution visualization of biomarker expression in vivo using superharmonic ultrasound imaging (SpHI) with dual-frequency transducers, targeted contrast agents, and localization microscopy processing. We validate and optimize the proposed method in vitro using concurrent optical and ultrasound microscopy and a microvessel phantom. With the same technique, we perform a proof-of-concept experiment in vivo in a rat fibrosarcoma model and create maps of biomarker expression co-registered with images of microvasculature. From these images, we measure a resolution of 23 µm, a nearly fivefold improvement in resolution compared to previous diffraction-limited molecular imaging studies.

Ultra high-frequency ultrasound with seventy-MHz transducer in hair disorders: Development of a novel noninvasive diagnostic methodology.

BACKGROUND: Ultra high-frequency ultrasound (uHFUS) is a recently developed diagnostic technology. Despite its potential usefulness, no study has assessed its advantage in diagnosis and evaluation of hair disorders in comparison with other diagnostic methods. OBJECTIVES: To assess the practicability of uHFUS in diagnosing hair disorders and propose a diagnostic methodology. METHODS: Ultrasonographic images of scalp and forehead from patients with hair disorders (n = 103) and healthy controls (n = 40) were obtained by uHFUS and analyzed by both descriptive and numerical parameters. Furthermore, the data were compared with trichoscopic and histopathological findings. RESULTS: The pattern of inflammation and fibrosis, hair cycle abnormality, and the findings in subcutis were detected by uHFUS. Significant differences were noted in the numerical parameters associated with the number of hair shafts and follicles, hair diameters and their diversity, and dermal echogenicity in both cicatricial and non-cicatricial hair disorders. Findings in uHFUS were associated with those observed in trichoscopy and scalp biopsy but uHFUS was able to detect pathological findings associated with hair cycle, inflammation, fibrosis, and subcutaneous abnormalities, which are hardly assessable by trichoscopy. CONCLUSION: The findings of this study highlighted usefulness of uHFUS in diagnosing hair disorders, while overcoming the weaknesses and limitations of other diagnostic tools.

Implementation of a Novel 288-Element Dual-Frequency Array for Acoustic Angiography: In Vitro and In Vivo Characterization.

Acoustic angiography is a superharmonic contrast-enhanced ultrasound imaging method that produces high-resolution, 3-D maps of the microvasculature. Previous acoustic angiography studies have used twoelement, annular,mechanicallyactuated transducers(called "wobblers") to image microvasculature in preclinical tumor models with high contrast-to-tissue ratio and resolution, but these earlywobbler transducerscould not achieve the depth and sensitivity required for clinical acoustic angiography. In this work, we present a system for performing acoustic angiography with a novel dual-frequency(DF) transducer-a coaxially stacked DF array (DFA). We evaluate the DFA system bothin vitro andin vivo and demonstrate improvements in sensitivity and imaging depth up to 13.1 dB and 10 mm, respectively, compared with previous wobbler probes.

A novel, hands-free ultrasound patch for continuous monitoring of quantitative Doppler in the carotid artery.

Quantitative Doppler ultrasound of the carotid artery has been proposed as an instantaneous surrogate for monitoring rapid changes in left ventricular output. Tracking immediate changes in the arterial Doppler spectrogram has value in acute care settings such as the emergency department, operating room and critical care units. We report a novel, hands-free, continuous-wave Doppler ultrasound patch that adheres to the neck and tracks Doppler blood flow metrics in the common carotid artery using an automated algorithm. String and blood-mimicking test objects demonstrated that changes in velocity were accurately measured using both manually and automatically traced Doppler velocity waveforms. In a small usability study with 22 volunteer users (17 clinical, 5 lay), all users were able to locate the carotid Doppler signal on a volunteer subject, and, in a subsequent survey, agreed that the device was easy to use. To illustrate potential clinical applications of the device, the Doppler ultrasound patch was used on a healthy volunteer undergoing a passive leg raise (PLR) as well as on a congestive heart failure patient at resting baseline. The wearable carotid Doppler patch holds promise because of its ease-of-use, velocity measurement accuracy, and ability to continuously record Doppler spectrograms over many cardiac and respiratory cycles.

Characterization of an Array-Based Dual-Frequency Transducer for Superharmonic Contrast Imaging.

Superharmonic imaging with dual-frequency imaging systems uses conventional low-frequency ultrasound transducers on transmit, and high-frequency transducers on receive to detect higher order harmonic signals from microbubble contrast agents, enabling high-contrast imaging while suppressing clutter from background tissues. Current dual-frequency imaging systems for superharmonic imaging have been used for visualizing tumor microvasculature, with single-element transducers for each of the low- and high-frequency components. However, the useful field of view is limited by the fixed focus of single-element transducers, while image frame rates are limited by the mechanical translation of the transducers. In this article, we introduce an array-based dual-frequency transducer, with low-frequency and high-frequency arrays integrated within the probe head, to overcome the limitations of single-channel dual-frequency probes. The purpose of this study is to evaluate the line-by-line high-frequency imaging and superharmonic imaging capabilities of the array-based dual-frequency probe for acoustic angiography applications in vitro and in vivo. We report center frequencies of 1.86 MHz and 20.3 MHz with -6 dB bandwidths of 1.2 MHz (1.2-2.4 MHz) and 14.5 MHz (13.3-27.8 MHz) for the low- and high-frequency arrays, respectively. With the proposed beamforming schemes, excitation pressure was found to range from 336 to 458 kPa at its azimuthal foci. This was sufficient to induce nonlinear scattering from microbubble contrast agents. Specifically, in vitro contrast channel phantom imaging and in vivo xenograft mouse tumor imaging by this probe with superharmonic imaging showed contrast-to-tissue ratio improvements of 17.7 and 16.2 dB, respectively, compared to line-by-line micro-ultrasound B-mode imaging.

30/80 MHz Bidirectional Dual-Frequency IVUS Feasibility Evaluated In Vivo and for Stent Imaging.

Although intravascular ultrasound (IVUS) is an important tool in guiding complex coronary interventions, the resolution of existing commercial IVUS devices is considerably poorer than that of optical coherence tomography. Dual-frequency IVUS (DF IVUS), incorporating a second, higher frequency transducer, has been proposed as a possible method of overcoming this limitation. Although preliminary studies have shown that DF IVUS can produce complementary images, including large-scale morphology and high detail of superficial features, it has not yet been determined that this approach would be feasible in a more clinically relevant environment. The purpose of this study was to demonstrate the first in vivo use of a 30/80 MHz DF IVUS catheter in visualizing coronary vessels in a porcine model. In addition, two commercially available stents were studied in vitro and in vivo. Clear subjective improvement of visualization of superficial structures is demonstrated, and sufficient dynamic range is achieved to image through both the catheter sheath and blood in vivo.

Superharmonic Ultrasound for Motion-Independent Localization Microscopy: Applications to Microvascular Imaging From Low to High Flow Rates.

Recent advances in high frame rate biomedical ultrasound have led to the development of ultrasound localization microscopy (ULM), a method of imaging microbubble (MB) contrast agents beyond the diffraction limit of conventional coherent imaging techniques. By localizing and tracking the positions of thousands of individual MBs, ultrahigh resolution vascular maps are generated which can be further analyzed to study disease. Isolating bubble echoes from tissue signal is a key requirement for super-resolution imaging which relies on the spatiotemporal separability and localization of the bubble signals. To date, this has been accomplished either during acquisition using contrast imaging sequences or post-beamforming by applying a spatiotemporal filter to the B-mode images. Superharmonic imaging (SHI) is another contrast imaging method that separates bubbles from tissue based on their strongly nonlinear acoustic properties. This approach is highly sensitive, and, unlike spatiotemporal filters, it does not require decorrelation of contrast agent signals. Since this superharmonic method does not rely on bubble velocity, it can detect completely stationary and moving bubbles alike. In this work, we apply SHI to ULM and demonstrate an average improvement in SNR of 10.3-dB in vitro when compared with the standard singular value decomposition filter approach and an increase in SNR at low flow ( [Formula: see text]/frame) from 5 to 16.5 dB. Additionally, we apply this method to imaging a rodent kidney in vivo and measure vessels as small as [Formula: see text] in diameter after motion correction.

Transcranial Photoacoustic Detection of Blood-Brain Barrier Disruption Following Focused Ultrasound-Mediated Nanoparticle Delivery.

PURPOSE: Blood-brain barrier disruption (BBBD) is of interest for treating neurodegenerative diseases and tumors by enhancing drug delivery. Focused ultrasound (FUS) is a powerful method to alleviate BBB challenges; however, the detection of BBB opening by non-invasive methods remains limited. The purpose of this work is to demonstrate that 3D transcranial color Doppler (3DCD) and photoacoustic imaging (PAI) combined with custom-made nanoparticle (NP)-mediated FUS delivery can detect BBBD in mice. PROCEDURES: We use MRI and stereotactic ultrasound-mediated BBBD to create and confirm four openings in the left hemisphere and inject intravenously indocyanine green (ICG) and three sizes (40 nm, 100 nm, and 240 nm in diameter) of fluorophore-labeled NPs. We use PAI and fluorescent imaging (FI) to assess the spatial distribution of ICG/NPs in tissues. RESULTS: A reversible 41 ± 12 % (n = 8) decrease in diameter of the left posterior cerebral artery (PCA) relative to the right after FUS treatment is found using CD images. The spectral unmixing of photoacoustic images of the in vivo (2 h post FUS), perfused, and ex vivo brain reveals a consistent distribution pattern of ICG and NPs at *FUS locations. Ex vivo spectrally unmixed photoacoustic images show that the opening width is, on average, 1.18 ± 0.12 mm and spread laterally 0.49 ± 0.05 mm which correlated well with the BBB opening locations on MR images. In vivo PAI confirms a deposit of NPs in tissues for hours and potentially days, is less sensitive to NPs of lower absorbance at a depth greater than 3 mm and too noisy with NPs above an absorbance of 85.4. FI correlates well with ex vivo PAI to a depth of 3 mm in tissues for small NPs and 4.74 mm for large NPs. CONCLUSIONS: 3DCD can monitor BBBD over time by detecting reversible anatomical changes in the PCA. In vivo 3DPAI at 15 MHz combined with circulating ICG and/or NPs with suitable properties can assess BBB opening 2 h post FUS.

Tumor Contrast Imaging with Gas Vesicles by Circumventing the Reticuloendothelial System.

Gas vesicles (GVs) are nanosized structures (45-800 nm) and have been reported to produce non-linear contrast signals, making them an attractive agent for molecular targeting of tumors. One barrier to their use for pre-clinical oncology studies is rapid uptake into the reticuloendothelial system (RES) and consequent rapid decrease in contrast signal after infusion ends and low signal on reperfusion after a bubble burst sequence. The purpose of this study was to examine suppression of the RES and surface modification of GVs to prolong contrast circulation in tumors for ultrasound imaging. Ultrasound imaging to measure dynamics of contrast signal intensity in tumor models was carried out using a 21-MHz high-frequency array transducer with the Vevo 2100 ultrasound system. The non-linear contrast signal from intravenously injected GVs compared with peak enhancement was measured during contrast wash-out and on reperfusion after a contrast burst sequence. Disrupting the RES by saturating the macrophage population or chemically inhibiting the Kupffer cell population with gadolinium or Intralipid preserves 62%-88% of GVs' contrast enhancement relative to peak during the wash-out phase and 32%-56% on reperfusion compared with 38% and 14%, respectively, for no disruption of the RES, indicating longer circulation of GVs in the tumor. Additionally, coating the GVs with 2-, 5- or 10-kDa polyethylene glycol (PEG) chains resulted in >70% contrast signal retention in the tumors during wash-out and, for 5- or 10-kDa PEG chains, a return to >45% of peak contrast signal on reperfusion. These findings indicate that GVs can be used as a contrast agent for tumor imaging and that disruption of the RES improved recirculation and maintained contrast enhancement caused by GVs in the tumors.

In Vitro Superharmonic Contrast Imaging Using a Hybrid Dual-Frequency Probe.

Superharmonic imaging is an ultrasound contrast imaging technique that differentiates microbubble echoes from tissue through detection of higher-order bubble harmonics in a broad frequency range well above the excitation frequency. Application of superharmonic imaging in three dimensions allows specific visualization of the tissue microvasculature with high resolution and contrast, a technique referred to as acoustic angiography. Because of the need to transmit and receive across a bandwidth that spans up to the fifth harmonic of the fundamental and higher, this imaging approach requires imaging probes comprising dedicated transducers for transmit and receive. In this work, we report on a new dual-frequency probe including two 1.7-MHz rectangular transducers positioned one on each side of a 20-MHz 256-element array. Finite element modeling-based design, fabrication processes and assembly of the transducer are described, as is integration with a high-frequency ultrasound imaging platform. Dual-frequency single-plane-wave imaging was performed with a microbubble contrast agent in flow phantoms and compared with conventional high-frequency B-mode imaging, and resolution and contrast-to-tissue ratio were quantified. This work represents an intermediate but informative step toward the development of dual-frequency imaging probes based on array technology, specifically designed for clinical applications of acoustic angiography.

High-Frequency Micro-Ultrasound Imaging and Optical Topographic Imaging for Spinal Surgery: Initial Experiences.

High frequency micro-ultrasound (µUS) transducers with central frequencies up to 50 MHz facilitate dynamic visualization of patient anatomy with minimal disruption of the surgical work flow. Micro-ultrasound improves spatial resolution over conventional ultrasound imaging from millimeter to micrometer, but compromises depth penetration. This trade-off is sufficient during an open surgery in which the bone is removed and theultrasound probe can be placed into the surgical cavity. By fusing µUS with pre-operative imaging and tracking the ultrasound probe intra-operatively using our optical topographic imaging technology, we can provide dynamic feedback during surgery, thus affecting clinical decision making. We present our initial experience using high-frequency µUS imaging during spinal procedures. Micro-ultrasound images were obtained in five spinal procedures. Medical rationale for use of µUS was provided for each patient. Surgical procedures were performed using the standard clinical practice with bone removal to facilitate real-time ultrasound imaging of the soft tissue. During surgery, the µUS probe was registered to the pre-operative computed tomography and magnetic resonance images. Images obtained comprised five spinal decompression surgeries (four tumor resections, one cystic synovial mass). Micro-ultrasound images obtained during spine surgery delineated exquisite detailing of the spinal anatomy including white matter and gray matter tracts and nerve roots and allowed accurate assessment of the extent of decompression/tumor resection. In conclusion, tracked µUS enables real-time imaging of the surgical cavity, conferring significant qualitative improvement over conventional ultrasound.

Ultrasound and Infrared-Based Imaging Modalities for Diagnosis and Management of Cutaneous Diseases.

Non-invasive bedside imaging tools are becoming more prevalent for assessing cutaneous lesions. Ultrasound used at specific frequencies allows us to assess margins of lesions to minimize the extent of the biopsy that is performed and improve cosmetic outcomes. Vascularity, seen on Doppler ultrasound and contrast-enhanced ultrasound, and stiffness, assessed on tissue elastography, can help differentiate between benign and malignant lesions for clinicians to be more judicious in deciding whether to biopsy. Moreover, research has shown the efficacy in using ultrasound in monitoring flares of hidradenitis suppurativa, a disease affecting apocrine gland-rich areas of the body, for which the current gold standard involves examining and scoring inflammatory lesions with the naked eye. Infrared-based modalities have also been on the uptrend to aid in clinical decision-making regarding suspiciousness of lesions. Reflectance confocal microscopy has lateral resolution that is comparable to histopathology and it has been shown to be an appropriate adjunctive tool to dermoscopy, specifically when evaluating melanomas. Optical coherence tomography has utility in determining lesion thickness because of its depth penetration, and spectrophotometric intracutaneous analysis is becoming more popular as a tool that can be used by general practitioners to know when to refer to dermatology regarding worrisome pigmented lesions. Strides have been made to incorporate electrical impedance spectroscopy alongside dermoscopy in decision-making regarding excision, although the evidence for its use in the clincial setting remains inconclusive. This paper reviews the efficacy and drawbacks of these techniques in the field of dermatology and suggests future directions.

In vivo Biodistribution of Radiolabeled Acoustic Protein Nanostructures.

PURPOSE: Contrast-enhanced ultrasound plays an expanding role in oncology, but its applicability to molecular imaging is hindered by a lack of nanoscale contrast agents that can reach targets outside the vasculature. Gas vesicles (GVs)-a unique class of gas-filled protein nanostructures-have recently been introduced as a promising new class of ultrasound contrast agents that can potentially access the extravascular space and be modified for molecular targeting. The purpose of the present study is to determine the quantitative biodistribution of GVs, which is critical for their development as imaging agents. PROCEDURES: We use a novel bioorthogonal radiolabeling strategy to prepare technetium-99m-radiolabeled ([99mTc])GVs in high radiochemical purity. We use single photon emission computed tomography (SPECT) and tissue counting to quantitatively assess GV biodistribution in mice. RESULTS: Twenty minutes following administration to mice, the SPECT biodistribution shows that 84 % of [99mTc]GVs are taken up by the reticuloendothelial system (RES) and 13 % are found in the gall bladder and duodenum. Quantitative tissue counting shows that the uptake (mean ± SEM % of injected dose/organ) is 0.6 ± 0.2 for the gall bladder, 46.2 ± 3.1 for the liver, 1.91 ± 0.16 for the lungs, and 1.3 ± 0.3 for the spleen. Fluorescence imaging confirmed the presence of GVs in RES. CONCLUSIONS: These results provide essential information for the development of GVs as targeted nanoscale imaging agents for ultrasound.

Development of a 3 French Dual-Frequency Intravascular Ultrasound Catheter.

Coronary plaque morphology, including plaque size and fibrous cap thickness, is thought to contribute to the risk of plaque rupture and future cardiac events. Dual-frequency intravascular ultrasound has been proposed as a possible technique to visualize both large-scale features and superficial detail of coronary plaque; however, it has not been found to be feasible within the constraints of a clinically functional intravascular ultrasound catheter. In this study, we describe the design and fabrication of a dual-frequency catheter using a bidirectional transducer stack with center frequencies of approximately 30 and 80 MHz. We describe how the high-frequency transducer achieves significantly improved axial and lateral resolution (16 and 120 µm, respectively, vs. 50 and 220 µm) at the expense of penetration depth. Finally, imaging of ex vivo human coronary artery segments reveals that the catheter can provide complementary images of the deeper arterial wall and superficial plaque features.